Since the discovery of the biological activity of short interfering RNAs (siRNAs) over a decade ago, so called “small RNAs” (i.e., short non-coding regulatory RNAs that have a defined sequence) have become a subject of intense interest in the research community. Exemplary short RNAs include siRNAs, microRNAs (miRNAs), tiny non-coding RNAs (tncRNAs) and small modulatory RNAs (smRNAs), as well as many others.
Although the exact biological fuinctions of most small RNAs remain a mystery, it is clear that they are abundant in plants and animals. For example, to date, over 78 Drosophila microRNA species and 200 human microRNA species have been identified. The levels of the individual species of small RNA, in particular microRNA species, appears to vary according to the developmental stage and type of tissue being examined. It is thought that the levels of particular small RNAs may be correlated with particular phenotypes, as well as with the levels of particular mRNAs and proteins. Further, viral microRNAs have been identified, and their presence has been linked to viral latency.
Methods that provide for quantitative detection of small RNAs are therefore extremely valuable. To this end, a considerable amount of effort is currently being put into developing array platforms to facilitate the analysis of small RNAs, particularly microRNAs.
However, in order to perform array experiments that provide quantitative results, it is highly desirable to use an array that contains probes that have similar melting temperatures (Tms) to each other. This presents a challenge for the design of arrays for the analysis of small RNAs because certain small RNAs are very short (e.g., in the range of 19-21 nucleotides in length), severely limiting the choices of sequences that can be employed in a probe for those small RNAs. Further, prior art small RNA detection methods may not discriminate between the small RNA and the precursor RNAs from which they are made, leading to results that do not accurately reflect the actual amount of a small RNA in a sample.
In view of the above, there is a great need for improved polynucleotide probes, particularly sets of polynucleotide probes having similar Tms, for assessing small RNAs in a sample. The invention described herein meets this need, and others.